Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
DNA hybridisation has been the most essential method for research in modern biological science involving the molecular studies of genes. The complexity of nucleic acid sequences of various genomes were revealed by solution hybridisation through the annealing process of the complementarity of specific DNA sequences. The annealing kinetics (Britten and Kohne, 1968) in solution of the complementary single strand DNA in solution is dependent upon the bimolecular collision. Thus the rate of reaction is directly controlled by the diffusion rate and the concentration of the nucleic acid target sequence. Although quantitative estimation of a single unique sequence by solution hybridisation has been very satisfactory, this technique can not be used for the study of multiple sequences in mixture unless elaborate separation process is done. This difficulty was eliminated by the discovery of membrane hybridisation techniques first developed by Southern (Southern E. M. 1975). The DNA in question was first digested with restriction endonucleases, sized by agarose gel electrophoresis and then immobilized onto a nitrocellulose membrane. The specific DNA sequence was identified by hybridisation with radioactively labelled complementary DNA probe. Since the same membrane can be used for reprobing through multiple repeat hybridisations with probes of different segment sequences, the genomic organisation of a specific gene or gene family can be obtained. Similar experiments can also be performed to gain further information on other sequences in the same membrane. The applicant and collaborators used allele specific oligonucleotide(ASO) to detect the point mutations of the Chinese population in Hong Kong (Antonarakis, S. E. et al., 1988). Thus the Southern Blotting analysis has been the most widely used molecular biology technique to date. In the so call ZOO BLOT (Miller J. R and Koopman M, 1990), in which a panel of Southern Blots consisting DNA from different sources, mapped with different restriction endonucleases and target DNA probes, one can establish the existence as well as the location of homologous sequences of a wide variety of spices within single membrane. If one simply want to know the existence of certain sequence, e.g. neither the size nor the genomic details, the dot and slot blot analysis can provide rapid screening procedure for a large number of samples. Unfortunately neither the Southern nor the dot or slot blot methods can be extended to the study of multiple sequences simultaneously within the same membrane in a single run e. g. without the time consuming procedures of repeat hybridisation by different probes. Furthermore the available membranes presently on the market for blotting analysis are generally unable to provide high affinity binding to small molecular weight polynucleotide fragments of less than 100 bp. The use of direct gel hybridisation reported by us (Huang C. H. et al., 1988) removed the blotting process thereby reduced the procedural time considerably. However DNA fragments of smaller than 300 bp are still not detectable. The reversed dot blot technique advanced by Maggio, A. et al (1993) provided the solution to these problems. In this method the a large number of capture oligonucleotides (20-24nt), used to capture the target DNA molecules, are covalently immobilized onto the Biodyne C membrane followed by hybridisation with the test sample(s) and colour development. With this immobilisation techniques the number of capture nucleotide is in principle limited only by the area of the membrane. Thus it is ideal for the detection of multiple mutations at the same point (base position) or at different points of the gene or even in different genes. This method has been applied for prenatal diagnosis of genetic diseases such as thalassaemia, haemophilia and other point mutations, including short insertion or deletions (Tam J. W. O. and Woo Y. K., 1994 and references therein). Thus the method provides an economical way to introduce to the rapidly advancing nucleic acid diagnostic market. However, as in the cases of Southern, dot or slot blotting, it is still a time and reagent consuming process. Due to the exponential growth of research activity and diagnostic development, demand for a even better hybridisation procedure is imperative. The present invention describes the principle of a unique hybridisation process and a device for the said purpose whereby the hybridisation time as well as the amount of reagents used for hybridisation can be reduced by many folds.
The principle of the present invention is using the direct flow-through mechanism by which the target Nucleic acid molecules pass through the membrane pore (the membrane porosity is about 0.45 micron with about 160 micron in thinness; As it will be well appreciated by a person of ordinary skill in the art that other sizes of pores and other thickness may be used in this invention.) allowing the single strand DNA of coming in close contact with the corresponding capture complementary DNA or RNA sequences immobilized inside the membrane pores so that the target sequence can be effectively detected in high sensitivity and specificity. At present when the conventional or the reversed Dot-blot was incubated either in a glass hybridisation tubes or plastic bags in water bath or hybridisation oven maintained at appropriate temperature, the time for complete hybridisation process requires several hours to several days. The reasons for the low rate of annealing process are: 1) The need for large volume to cover the whole of the membrane is necessary. Consequently the concentration of the probe in solution available for binding the target DNA is low. Since the annealing kinetic is a bimolecular collision process, such a dilution will have enormous effect on the rate for the probe to find its target complement on the membrane; 2) During the incubation process, the majority of the solution does not make contact with the membrane. This will increase the chance of the self-annealing process of the otherwise separated complementary strands (since most of the probe or test DNA are double-stranded in origin which were denatured by heating to become single strands) DNA probe. Consequently the effective concentration of the probe is reduced further. The longer hybridisation time the lower the concentration of hybridisable single strand DNA will become resulting in still lower rate of hybridisation kinetic; and 3) In the immersion hybridisation process the most accessible target DNA molecules for probe binding are those immobilized on the surface of the membrane. Unfortunately only a small part of target DNA is actually directly facing the outside because of the intrinsic characteristics of the membrane being porous (generally from 0.1 to 0.45 micron in diameter) and therefore during the immobilized process a large part of the DNA will be immobilized in the interior part of the membrane (that is why in the blotting process smaller fragments, &lt;500 bp for 0.45 micron and &lt;100 bp for 0.1 micron membranes, diffuse through the membrane and lost). The rate of annealing of these target DNA will then be slowed down further by the slow diffusion process of the probe into the inside pores of the membrane. The result would be a decrease in sensitivity even with prolong incubation. A flow through hybridisation process described in the present invention eliminates all of the above intrinsic defects.
Recently a flow through hybridisation assay for oligonucleotide sequences described by the European Patent application (application number: 93,120,394.7; Publication number:605828 A1) by Felndt, H. H., Mallonee R. L. and McFarland E. C of Becton, Dickinson & Co used the immunological assay flow-through methods described in earlier arts (U.S. Pat. Nos. 4,366,241; 4,727,019; 4,632,901 and 4,818,677) for the detection of DNA sequences. However, this method did not have the controlled stringency elements and therefore can only be used to discriminate non-homologous sequences (even with this, there appear to produce high background noise as stated in Example 3 of document EPO 605 828 A1, page 7 line 15). It cannot achieve the required specificity and sensitivity for stringent detection even grossly related sequences. The most crucial requirement for nucleic acid hybridisation in achieving high specificity without scarifying sensitivity is the accurate control of stringency. This requirement is a must especially when discrimination amongst closely related sequences where homology is very high e.g. allelic variants, family of genes within the same organism and similar sequences from different organisms. Examples of diagnosis of genetic diseases such as the non-deletion types of thalassaemia of human beta globin genes or infectious agents like variants of HCV, HIV and others. Even in the cases of differentiating between family of genes of similar function in different organisms the flow-through process described by the prior art of Felndt et al. cannot produce the satisfactory results (see Example V of this document in later section below). The present invention discloses the methods and the apparatus necessary to achieve the most important controlled conditions for rapid and specific nucleic acid hybridisation that has never been addressed before in prior arts. The novelty of the present invention is that it can provide the controlled conditions critical for the hybridisation process which can be universally applied to all cases that is applicable to conventional Southern, Northern, Dot-Blot, Slot-Blot and Reversed-Dot Blot hybridisation techniques so far reported on the literature. When it is used for Western Blotting (protein blotting or immunoblotting), improvement on sensitivity and specificity will be expected because the reaction conditions can be accurately controlled.